T lymphocytes play critical roles in immune defense against viruses, bacteria, fungi, protozoa, and cancer cells. Upon encounter with antigens on specialized presenting cells (dendritic cells), these resting T-cells differentiate into effector cells that leave the lymphoid tissues and blood, entering sites of infection to combat pathogens. They can also cause autoimmune pathology. Most activated T-cells die, but some remain as memory cells, with some in lymphoid compartments and others patroling peripheral tissues. Other lymphocytes such as regulatory T cells contribute to suppression of these T-cell responses. T cells also play a central role in the regulation of B cell antibody responses. This project attempts to gain both a qualitative and quantitative understanding of the activation, differentiation, migration, cell-cell interaction, memory status, and reactivation properties of both CD4 and CD8 T-cells and their interactions with other immune cells types such as dendritic cells and B cells. Through this research, a better understanding of lymphocyte dynamics and tissue architecture during an immune response to infection or after vaccination or during an autoimmune response will be established. These new insights can contribute to the more effective design of vaccines and to strategies for the amelioration of autoimmune processes. In a project related to studies described in ZIA AI000545-32, we previously used Histo-cytometry to discover that pSTAT5+ Treg cluster around mostly migratory dendritic cells together with Tconv cells. Strikingly, the same pSTAT5+ Treg clusters are seen at comparable numbers in germ-free and conventional SPF mice, indicating that the activation of Tconv to the IL-2 producing state involves self-recognition. Building on these seminal observations, we are now in the midst of studies to determine the fate of the auto-activated CD4 T cells whose potential for tissue damage is controlled by the co-clustering Treg. Initial results indicate that the activated Tconv cells express PD-1 and many are associated with activated caspase, suggesting post-activation induced cell death as a possible fate. The accumulating data suggest an intricate, non-linear combination of factors involving Treg limitation of co-stimulatory signals, consumption of IL-2, disruption of CD25 upregulation, and cytokine deprivation may underpin the way Tregs protect immune homeostasis. These studies have been markedly accelerated and made more informative through application of novel methods of statistical analysis of cell distribution in tissues using techniques such as paired correlation analysis, as well as through use of C3eD tissue clearing (see ZIA AI000545-32 LSB), which has permitted us to examine physiologically small numbers of cells in entire lymph nodes cleared, stained and imaged using this methodology. Using Histo-cytometry, we have discovered that in contrast to the assumption that CD4+ and CD8+ T cells are randomly mixed within the paracortical T cell area of lymph nodes, there is a skew in the CD4+ T cell distribution that matches our previously described asymmetric distribution of cDC1 and cDC2 cells with their bias for MHC class I vs. MHC class II antigen presentation. This pattern is dictated by the chemoattractant receptor Ebi2 and disturbance of this distribution negatively impacts protection against helminth infection or protection post-vaccination against liver stage malaria. Mechanistic studies indicate that the loss of protection is due to a limitation of CD4+ T cell help for CD8+ memory T cell responses, consistent with the idea that preventing the local pre-concentration of CD4 T cells near their relevant presenting cDC2s lengthens the time it takes for the CD4 T cells to find a useful antigen-bearing DC and hence, disrupts efficient delivery of help to already activated CD8 T cells. These findings extend our previous emphasis on the critical role played by tissue micro-anatomy in the effective operation of the innate and adaptive immune systems. We have now employed Histo-cytometry to study human immune responses in tissues rather than as isolated blood cells. In a collaboration with D. Canaday from Case Western Reserve, we used multiplex immunohistochemistry to examine the distribution of T follicular regulatory cells (Tfr) in mesenteric lymph nodes removed during abdominal surgery. This research revealed that in contrast to the prevailing view that Tfr worked by interfering with the germinal center reaction within that specialized structure, the vast majority of Tfr were outside the germinal center in the larger follicular area. These data indicate that models of Tfr function need to be revised to comport with these new topographic data that suggest action within the follicle but outside the germinal enter proper. Tfr may compete for necessary antigen-specific interactions at the GC border, preventing entry of Tfh. This study is one of the few that draws functional interpretations of immune action in human tissues based on highly multiplex histochemical analysis combined with more conventional tools such as in vitro culture and flow cytometry. To gain a deeper understanding of how T cells acquire their selective differentiated phenotypes, we have begun a new effort to improve our capacity of long term (6-12 hour) 2P IVM of lymphocytes and dendritic cells within lymph nodes. Our initial focus will be on the so-called Phase 3 of T- cell dynamic behavior during initial priming. This period follows initial search for antigen-bearing DCs (Phase 1), and prolonged interactions without substantial migration between T cells and DCs (Phase 2). Little is known about Phase 3, during which there is rapid migration of the T cell blasts generated in Phase 2. Whether there are productive, if nonetheless short, interactions of the T cells with antigen bearing DCs, how expression of activation associated molecules that dampen immune signaling, such as PD-1, which are now present on the membranes of the activated T cells, affects fate choice, and are there topographic features of where distinct types of T cell development occur within a lymph node are all critical issues to be resolved in such studies. Beyond improving the IVM aspects of the analysis, we are also seeking to develop a robust method to taking the lymph nodes that have been live imaged and using Histo-cytometry and Ce3D imaging to relate the dense phenotype of T cells to their prior dynamic behavior. We believe this combination of new technology and its application to an underexplored aspect of T cell immune activation will provide important insights into how distinct subsets of T cells develop, with implications for understanding autoimmune diseases and for vaccine design. To better understand the signaling events involved in T cell activation, the shaping of the effector and memory repertoire, and the function of T cells exposed in a chronic manner to antigen (for example, certain viral infections or cancer), we have developed an entirely new approach that combines multiplex live reporters with single cell dynamic imaging in vitro. This approach allows us to manipulate the genotype of the T cells using CRISPR technology (eliminating or forcing expressing), while monitoring the signaling response at multiple levels (p65 NFkB, ERK, p38, JNK, cRel, NFAT) in real time in response to TCR engagement by strong and weak ligands in the presence or absence of PD-1/PD-L1 and/or CD28/CD80 engagement. Our initial results have revealed a clear dichotomy in the triggering of certain signaling pathways by TCR, CD28, or the combination, and suggested that the emerging view of PD-1 action primarily via CD28 inhibition is too limited a model for what happens in the face of varying antigen density and quality in complex systems such as a tumor.